Association between follicular fluid bacteria with inflammatory markers of the complete blood count and the outcomes of assisted reproductive technology in women with endometriosis: A case–control study

Abstract Background and Aims Endometriosis is a common reason for infertility and poor outcomes of assisted reproductive technology (ART). Inflammation is involved in the pathogenesis of this disease. The presence of microorganisms in women with endometriosis may increase levels of inflammatory markers. The purpose of this study is to determine the relationship between the presence of bacteria in the follicular fluid with the inflammatory markers of the complete blood count (CBC) and the outcomes of in vitro fertilization (IVF) in women with endometriosis. Methods This case–control study was conducted on 74 patients undergoing IVF, referred to Al‐Zahra Hospital in Rasht (Iran) in 2021. The patients were divided into two case groups including 37 women with endometrioma and the control group, including 37 infertile women with a male factor and normal ultrasound. In total, 74 follicular fluids were collected from the case and control groups and were cultured in the laboratory. The relationship between culture results with IVF outcomes and the levels of CBC inflammatory markers including the number of white blood cells (WBCs), lymphocytes, neutrophils, neutrophil‐to‐lymphocyte ratio (NLR), lymphocyte‐to‐monocyte ratio (LMR), and platelet‐to‐lymphocyte ratio (PLR), erythrocyte sedimentation rate (ESR), and c‐reactive protein (CRP) was analyzed. Results There was no significant statistical difference between the frequency of bacteria present in the follicular fluid (p = 0.861), the mean rate of fertilization (p = 0.363), the frequency of CRP (p = 0.999), and the mean WBCs, lymphocytes, neutrophils, NLR, LMR, and PLR in the two groups. There was a significant statistical difference between the mean number of oocytes of metaphase II (p = 0.034) and the mean ESR (p = 0.018) in the two groups. Conclusions It seems necessary to evaluate follicular fluid as a biological substance that is considered an optimal factor for predicting oocyte quality, fertilization rate, embryo quality, and the success rate of ART.

Methods: This case-control study was conducted on 74 patients undergoing IVF, referred to Al-Zahra Hospital in Rasht (Iran) in 2021.The patients were divided into two case groups including 37 women with endometrioma and the control group, including 37 infertile women with a male factor and normal ultrasound.In total, 74 follicular fluids were collected from the case and control groups and were cultured in the laboratory.The relationship between culture results with IVF outcomes and the levels of CBC inflammatory markers including the number of white blood cells (WBCs), lymphocytes, neutrophils, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR), erythrocyte sedimentation rate (ESR), and c-reactive protein (CRP) was analyzed.
Results: There was no significant statistical difference between the frequency of bacteria present in the follicular fluid (p = 0.861), the mean rate of fertilization (p = 0.363), the frequency of CRP (p = 0.999), and the mean WBCs, lymphocytes, neutrophils, NLR, LMR, and PLR in the two groups.There was a significant statistical difference between the mean number of oocytes of metaphase II (p = 0.034) and the mean ESR (p = 0.018) in the two groups.

| INTRODUCTION
Endometriosis is a chronic inflammatory disease characterized by the growth of endometrial tissue outside the uterine cavity and affects 10% of reproductive-aged women. 1,2Endometriosis may extend to the ovaries and result in the formation of an endometrioma cyst. 2,3Approximately 17%-44% of women diagnosed with endometriosis have an endometrioma. 3Endometriosis is a common cause of infertility.About 25%-50% of infertile women have endometriosis, and 30%-50% of women with endometriosis are infertile. 2cording to studies, endometriosis as an inflammatory disease is associated with dysfunction of immune cells, especially macrophages, and abnormal and excessive production and secretion of immune mediators such as cytokines, prostaglandins, and metalloproteases. 4,5In other words, in women with endometriosis, ectopic endometrial tissue increases the number of macrophages in the peritoneal cavity by releasing cytokines and chemokines.An increase in the macrophages may also increase the inflammatory factors such as monocyte chemotactic protein-1, tumor necrosis factor-α, interleukin-8 (IL-8), and IL-6 and result in the worsening of the inflammation. 6,7An increase in proinflammation factors such as IL-6 may also result in an increase of other inflammation markers such as c-reactive protein (CRP). 8[11] Assisted reproductive technology (ART), especially in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI), is one of the most widely used treatment strategies in infertile women with endometriosis. 3 Follicular fluid plays an important role in steroidogenesis, follicle growth, oocyte maturation, ovulation, and oocyte transport, and is an optimal factor for predicting oocyte quality, fertilization rate, embryo quality, and IVF/ICSI success. 12,137][18] In other words, microbial colonization of the upper genital tract is one of the independent factors that influence the success rate of ART. 19According to a research conducted to evaluate the effect of microbial colonization of follicular fluid on the outcomes of ART, bacterial colonization in follicular fluid was associated with a decrease in fertilization rate and viable embryos in ART cycles, especially in women with endometriosis. 17dometriosis is associated with inflammation, so 4,20 the systemic inflammatory response can be associated with changes in different categories of white blood cells (WBCs). 20The neutrophil-tolymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in peripheral blood are considered as simple indicators of systemic inflammatory response and disease prognosis. 20,21According to a research that was done to assess the diagnostic value of NLR alone and combined with CA-125 for the diagnosis of endometriosis, the mean NLR and the combined marker of NLR and CA-125 in patients with endometriosis were significantly higher than patients without endometriosis.In this study, NLR was also able to distinguish patients with endometriosis from patients with benign ovarian tumors and healthy people. 20udies suggest that prescribing broad-spectrum antibiotics may be effective in treating human endometriosis by reducing inflammation, cell proliferation, and angiogenesis. 22,23Vaginal antibiotics may help treat endometriosis, according to a study in animal models, but more research is needed before the results can be generalized to humans. 24 one of the clinical predictors of inflammation diagnosis is the complete blood count (CBC)-based biomarkers and this evaluation is an available cost-effective method, the purpose of this study is to determine the relationship between the presence of bacteria in follicular fluid with the inflammatory markers of the CBC and the outcomes of IVF in women with endometriosis.

| Study design and setting
The present research was a case-control study that was performed to determine the relationship between the presence of bacteria in follicular fluid with the inflammatory markers of the CBC and the outcomes of IVF in women with endometriosis who were referred to the infertility clinic of Al-Zahra Hospital in Rasht (Iran) in 2021-2022.This case-control study was conducted between November 2021 and October 2022.Participants were 74 women aged 18-40 undergoing IVF.The sample size was determined based on a confidence interval (CI) of 90% and power of 80%, and after 10% attrition, the number of participants in each group was determined to be 37.The participants were divided into case group including 37 women with endometriosis identified by ultrasound (presence of endometrioma) and the control group, including 37 infertile women with a male factor and normal ultrasound and no signs of the presence of endometriosis or other infertility-related diseases.Both case and control groups were matched in terms of age, body mass index (BMI), and ovarian stimulation protocol.The exclusion criteria include patients who have not responded to ovarian stimulation and recent antibiotic use within the past 3 months.
The participants were included in the study by simple sampling method.IVF treatment started for both groups after informed consent.
To evaluate follicular fluid and perform IVF, 2 mg of estradiol was prescribed twice a day from the 20th day of the previous cycle.On the second day of the cycle, ultrasound was performed, and if there was no follicle above 10 mm, ovarian stimulation was induced by follicle-stimulating hormone (Cinnal-F, CinnaGen)/human menopausal gonadotropin (PD HOMOG).The ovarian response to the stimulation was evaluated by ultrasound.The first ultrasound was performed 5-6 days after ovulation stimulation.If there were 13-14 mm follicles in the ultrasound, gonadotropin-releasing hormone antagonist (Cetreotide, 0.25 mg; Merck) was prescribed to prevent luteinizing hormone surge.The ovarian stimulation was stopped after observing at least two 18 mm follicles in ultrasound, and 10,000 U of human chorionic gonadotropin (hCG) was injected.About 36-40 h after administration of hCG, ovarian follicles were aspirated under anesthesia and using a vaginal ultrasound guide.
After IVF, the embryos were classified into four A-D grades based on quality.
In this study, the follicular fluid was aspirated under sterile conditions and sent to the laboratory to detect the presence of bacteria in follicular fluid from the largest and most available follicles (17-20 mm) in the left or right ovary.The result of the culture was the number of bacterial colonies and the type of bacteria (Grampositive or Gram-negative).

| Data analysis
The research was conducted using SPSS version 21.The normality test of continuous variables was done by the Shapiro−Wilk test.
Normally distributed continuous variables were described by mean and standard deviation; nonnormally distributed continuous variables were described by median and interquartile range (IQR); categorical variables were described by count and percentage.To compare the means of two-category variables, the independentsamples t-test was used.The Mann−Whitney U-test was utilized for nonparametric comparisons, such as the mean number of oocytes, the mean rate of fertilization, and the mean inflammatory markers between the two groups.To measure the relationship between qualitative variables, the χ 2 test was used.All the above analyses were considered statistically significant with p < 0.05.

| RESULTS
The participants in this study were 74 women between the ages of 18 and 40 who had undergone IVF, and none of them were excluded from the study.Based on the findings of this study, the mean age of women with and without endometriosis was 35.38 ± 4.79 and 35.84 ± 5.52, respectively.There was no significant statistical difference between the mean ages of the two groups (p = 0.566).
The mean BMI in women with and without endometriosis was 26.98 ± 2.63 and 25.67 ± 2.47, respectively.There was no significant statistical difference between the mean BMI of the two groups (p = 0.051).
The presence of bacteria in follicular fluid in the endometriosis and nonendometriosis groups included 21 (56.7%) and 32 (59.5%) cases, respectively, and all the bacteria were Gram-positive in both groups.There was no significant statistical difference between the frequencies of bacteria present in the follicular fluid of the two groups (p = 0.861).Additional information is inserted in Table 1.
The mean number of metaphase II (M II) oocytes in women with and without endometriosis was 4.22 ± 3.58 and 6.51 ± 4.79, respectively.There was a significant statistical difference between the mean numbers of M II oocytes of the two groups (p = 0.034).
The mean fertilization rate of women with and without endometriosis was 61.73 ± 34.22 and 68.35 ± 34.56.There was no significant statistical difference between the mean fertilization rates of the two groups (p = 0.363).
T A B L E 1 Distribution of frequency of the bacteria present in follicular fluid of the two groups of women with and without endometriosis.The distribution of the mean quality of the embryo in grades A-D in both groups of women with or without endometriosis is included in Table 2.There was no significant statistical difference between the mean embryo qualities in grades A, B, C, and D in the two groups.
The distribution of mean blood inflammatory markers in the two groups of women with and without endometriosis is inserted in Table 3.There was no significant statistical difference between the mean WBC, lymphocytes, neutrophils, NLR, LMR, and PLR in the two groups.
The CRP frequency in the groups of women with and without endometriosis was two persons (5.4%) and one person (2.7%) respectively.There was no significant statistical difference between the CRP frequencies of the two groups (p = 0.999).
The mean ESR in women with and without endometriosis was 20.48 ± 14.05 and 15.81 ± 10.91, respectively.There was a significant statistical difference between the ESR frequencies of the two groups (p = 0.018).

| DISCUSSION
The present study was conducted to investigate the relationship between the presence of bacteria in follicular fluid with the inflammatory markers of the CBC and the outcomes of IVF in women with endometriosis.In line with the results of this study, based on a study that was performed to compare the bacterial patterns present in endometriotic lesions, endometrium, and vaginal discharge from women with and without endometriosis, no significant differences were found in the bacteria profiles between control and endometriotic patients. 25However, based on another study that aimed at molecular detection of microbial colonization in the cervical mucus of women with and without endometriosis, Streptococcus and Enterobacteriaceae in cervical mucus were more frequently detected in women with endometriosis than the women without it. 26cording to previous studies, the presence of various microorganisms in follicular fluid and secreted cytokines from them may decrease the pregnancy rate and have a negative effect on the IVF/ICSI outcomes, especially in women with endometriosis. 17,27,28Based on a study that aimed to investigate the influence of the microbial colonization of follicular fluid on ART outcomes, bacterial colonization in follicular fluid was associated with a decrease in the fertility rate and viable embryos in ART cycles, especially in women with endometriosis. 17Contradictorily, based on another study that aimed to investigate the effect of microorganisms present in follicular fluid on IVF outcomes, bacterial colonization of follicular fluid had no effect on the number of retrieved oocytes, fertilization and pregnancy rates, and IVF outcomes. 29Based on another research that was performed to identify and compare the microorganisms present in follicular fluid and their influence on IVF/ICSI outcomes, there was no relationship between the microorganisms present in follicular fluid and the embryo quality and the rate of pregnancy in IVF/ICSI cycles. 14 the interpretation of the above contradictory findings, it is important to point out that currently the effect of bacteria on the oocyte is not well known and there are relatively few studies have been performed on the microorganisms present in the follicular fluid that might play a potential role in women's fertility. 27cording to the findings of the present study, the mean number of M II oocytes in women with and without endometriosis was 3.58 ± 4.22 and 4.79 ± 6.51, respectively, and there was a significant statistical difference between the mean numbers of M II oocytes of the two groups of women with and without endometriosis (p = 0.034).There was no significant statistical difference between the mean fertilization rate and grades A, B, C, and D embryo qualities of the two groups of women with and without endometriosis.
T A B L E 2 Distribution of the mean embryo quality in the two groups of women with and without endometriosis.In line with the results of the present study regarding the mean number of M II oocytes, the fertilization rate, and embryo quality, based on a meta-analysis study, although endometrioma was associated with a decrease in the number of M II oocytes retrieved in IVF/ICSI cycles, it had no negative influence on the total number of embryos and also on the number of high-quality embryos. 3According to another research that aimed to study the impact of endometrioma cysts on the morphology of oocytes and the fertility outcomes in ICSI cycles, although M II oocytes were fewer in patients with endometrioma, there was no significant statistical difference between the fertility rates of the two groups. 30Contrary to the findings of the present research, based on one study that aimed to compare the IVF outcomes in women with and without endometriosis who have decreased ovarian reserves, endometrioma has no influence on the number of retrieved oocytes and the IVF outcomes such as implantation and the rate of live birth. 31 interpret the above-mentioned contradictory findings, it should be noted that endometriosis is an estrogen-dependent chronic inflammatory disease and its pathogenesis is multifactorial in causing infertility. 1 The toxic content of endometrioma cysts such as inflammatory mediators, iron, and reactive oxygen species may influence the ovarian and folliculogenesis reserves by inflammation and destruction of the ovarian cortex and result in the qualitative and quantitative decline in oocytes and therefore poor quality of embryos. 10,31,32Ovarian reserve is an important factor in evaluating the success rate of ovulation induction and ART. 33e of the clinical predictors of inflammation detection is CBCbased biomarkers.In the present study, this predictor included studying the number of cells and their ratio.Based on the findings of the present research, there was no significant statistical difference between the mean WBC, lymphocytes, neutrophils, NLR, LMR, and PLR in the two groups of women with and without endometrioses.
There was no significant statistical difference between the frequency of CRP in the two groups (p = 0.999), but there was a significant statistical difference between the mean ESR in the two groups (p = 0.018).
In line with the findings of the present study, based on a study that aimed to evaluate the mean platelet volume (MPV), NLR, and PLR in advanced endometriosis with endometrioma, the participants included 94 patients who were divided into three groups that consisted of 33 patients with endometrioma, 28 patients with endometriosis who did not have endometrioma, and 33 patients who underwent laparoscopic tubal ligation.According to the findings of this research, there was no significant statistical difference in the MPV, NLR, and PLR of the three groups. 21Based on a study that was performed to evaluate the role of inflammatory markers in detecting extraperitoneal endometriosis, laboratory assessment included the study of CA-125, thyroid-stimulating hormone, CBC differential T A B L E 3 Distribution of mean blood inflammatory markers in the two groups of women with and without endometriosis.| of 8 count, MPV, monocyte-to-platelet ratio, NLR, LMR, and PLR.In this research, no significant statistical difference was observed in the routine laboratory outcomes of the patients and inflammatory markers.Besides, there was no significant relationship between the inflammatory marker levels in patients who underwent surgery because of endometriosis in different locations with various symptoms. 34ntrary to the findings of the present study, based on a study that aimed to investigate the diagnostic value of combination markers of NLR and CA-125 for differentiating endometrioma from other benign ovarian cysts, the NLR and CA-125 levels were assessed separately and combined.Based on the findings of this research, neutrophils, lymphocytes, and NLR were higher in women with endometriosis than in women without it.There was also a direct relationship between the stage of the disease and the size of the endometrioma and the rate of NLR increase. 35 explain these findings, we may say that although the NLR and PLR in peripheral blood are considered simple markers of systematic inflammatory reactions and disease prognosis and assessing these parameters in the peripheral blood is an available and cost-effective method, 20,21,34 more studies are needed in this regard.

| STRENGTHS AND LIMITATIONS
One of the strengths of this study was to evaluate the follicular fluid as an optimal factor in predicting the oocyte quality, fertilization rate, embryo quality, and success rate of ART.Besides, assessing the inflammatory markers of CBC is an available, noninvasive, diagnostic, and cost-effective method of detecting infection in women with endometriosis and this may provide a valuable context for further future studies.One of the limitations of the present study was that since few studies have been performed on the CBC-based biomarkers as a clinical predictor of infection diagnosis in women with endometriosis.Therefore, further comparison of the results was not possible.Another limitation of this study is procedural bias.In this study, we attempted to aspirate follicular fluid under completely sterile conditions, but the sample may have been contaminated during aspiration and interfered with the interpretation of the results.

| CONCLUSIONS
There was no significant statistical difference between the frequency of bacteria present in the follicular fluid in the two groups of women with and without endometrioses who were undergoing IVF.There was a significant statistical difference between the mean numbers of oocytes of M II in the two groups.There was no significant statistical relationship between the fertilization rate and grades A, B, C, and D embryo qualities in the two groups.There was no significant statistical difference between the mean WBC, lymphocytes, neutrophils, NLR, LMR, and PLR in the two groups.In our study, there was no statistically significant difference between the frequency of CRP in the two groups, but the mean ESR showed a statistically significant difference.It seems necessary to evaluate follicular fluid as a biological substance that is considered an optimal factor for predicting oocyte quality, fertilization rate, embryo quality, and the success rate of ART.